ABSTRACT Influenza A viruses pose a serious threat to human health, causing seasonal epidemics and occasional pandemics that result in significant morbidity and mortality worldwide. The virulence and pathogenic nature of influenza virus strains vary greatly, and is often determined by the constellation of viral genes. While seasonal influenza viruses cause a mild upper respiratory infection in healthy individuals, highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype replicate in the lower respiratory tract, causing fatal viral pneumonia characterized by pulmonary edema and vascular leakage. In addition, H5N1 infections are associated with an uncontrolled activation of host immune responses, which contributes to the severity of disease. The role of viral and host factors in the enhanced virulence and pulmonary immunopathology characteristic of HPAIV infections remain unknown. To this end, we engineered H5N1 viruses with restricted tropism through the incorporation of microRNA (miRNA) target sites into the viral genome. Specifically, we generated an H5N1 virus carrying endothelial cell specific miR-126 target sites (H5N1-126T), such that viral replication was abrogated in endothelial cells without impeding replication in other cell types. In our recent studies, H5N1-126T infected mice and ferrets showed significantly reduced virulence and vascular leakage in the lungs as compared to the control H5N1-ScrbT virus infected group, despite similar viral loads in the lungs of mice and nasal washes of ferrets. To our knowledge, this is the first study to demonstrate the importance of endothelial cell tropism to H5N1 pathogenesis. The current proposal will (1) determine the consequence of endothelial cell infection to barrier integrity and function, (2) investigate the role of endothelial cell tropism in orchestrating immune responses, and (3) assess the contribution of viral tropism to the pathogenesis of other highly virulent strains including H7N7, 1918 H1N1, and H7N9 in mice and ferrets. The knowledge gained from these studies will help to elucidate the mechanism by which virulent influenza viruses cause severe disease and aid in the development of novel therapeutic strategies against virulent influenza strains. Furthermore, these studies will reveal how viral tropism modulates host antiviral responses and will pave the way for the rational design of novel live attenuated vaccines with restricted tropism.